This application directly responds to several objectives outlined in the RFA NR15-001. Specifically, it addresses the role of bacterial colonization of chronic wounds; impact of bioburden on repair process; mechanisms associated with chronic wounds. The objective of this project is to determine the role of antimicrobial molecule, Perforin-2 (P-2) in wound infection and wound healing and to optimize P-2 levels to either prevent or more rapidly resolve bacterial wound infections and speed healing. Wound infection is one of the most frequent complications representing one of the leading causes of lower leg amputations in patients suffering from chronic non-healing wounds, such as diabetic foot ulcers (DFUs).Thus, there is an urgent need to develop new approaches that prevent and treat infected wounds, especially those caused by antibiotic- resistant microorganisms. Our preliminary data suggest that skin cells are equipped to fight infections by expressing high levels of P-2, an antimicrobial protein with unique properties to kill intracellular bacteria. Also, down-regulation of P-2 in healing impaired wounds may contribute to chronic infections and inhibition of healing. We hypothesize that P-2 is necessary for the wound healing process, i.e. barrier maintenance and restoration and further, that the aberrant down-regulation of P-2 plays a role in infection propagation and healing inhibition. To test this we will: assess differential expression f P-2 in DFUs and correlate P-2 expression with healing outcomes (Aim 1); Determine how presence or absence of P-2 in primary HEK and dermal fibroblasts HDF grown in low and high glucose concentration, thus mimicking DFU environment, will affect their ability to kill intracellular bacteria. (Aim 2); identify the role of P-2 in acute human wound healing. (Aim 3). This project will provide important data and new knowledge towards understanding mechanisms by which P-2 expression contributes to protection from infection and P-2's role in non-healing wounds (DFUs). This project may provide the basis for development of novel modes for prevention of infection or treatment regimens and potentially simultaneously accelerate wound healing, both of which would have a major clinical impact and significance in both the acute and chronic wound infections. We anticipate that successful completion of this project will lead to future studies to develop candidate therapeutics through preclinical and clinical testing.